My long-term goal is to understand the cellular mechanisms of intestinal lipid uptake. My work focuses on developing tools to exploit the optical clarity of the zebrafish embryo, a feature that enables visualization of lipid uptake in live animals. The aims of this proposal are: (1) Visualize intestinal lipid absorption in live zebrafish larvae. I will develop methods for feeding larval zebrafish a high-fat diet and imaging intestinal lipid absorption. Experiments proposed will determine the rates of LA formation and utilization in live enterocytes using specific lipophilic fluorescent dyes coupled with confocal microscopy. (2) Determine the subcelluar organelles/proteins involved in enterocyte lipid absorption. It is unknown which organelles are used by enterocytes to sort the large amounts of mixed lipids taken in during absorption. By examining the formation of LAs over time, determine if LAs form from ER that is localized near the apical enterocyte compartment Create fluorescent fusion proteins, to determine if and when ER co-localizes with LDs produced following feeding. (3) Determine if LAs utilize a Niemann-Pick C1 Like 1 (Npc1l1)/ezetimibe pathway. Ezetimibe is a drug currently used to treat human hypercholesterolemia and hypothesized to target intestinal NPC1I1. However the timing, location of NPC1L1, and subcellular changes in lipid localization/trafficking that occur during ezetimibe treatment are poorly characterized. A human NPC1L1-YFP transgene will be used to test the hypothesis that feeding alters NPC1L1 localization. This Aim will clarify ezetimibe's mechanism of action and NPCIH's role in lipid absorption. Obesity in the U.S. has reached epidemic levels and will afflict 41% of Americans by 2015. PUBLIC HEALTH RELEVANCE: Obesity-related diseases such as coronary heart disease and type-2 diabetes are leading killers in the U.S. Understanding the mechanisms involved in how the vertebrate intestine absorbs lipids and how small molecules can modulate these processes will help provide insights into obesity-associated diseases and their treatments.